摘要
目的·研究SUMO化修饰对胸腺嘧啶DNA糖基化酶(thymine DNA glycosylase,TDG)蛋白的结构、稳定性及活性的影响。方法·建立体外表达纯化体系,获取可用于晶体筛选及活性检测的SUMO-1-TDG蛋白。通过晶体筛选、衍射数据收集及结构解析,分析SUMO-1-TDG的分子结构。利用蛋白热稳定性实验检测在SUMO化修饰前后TDG稳定性的变化。建立TDG活性测试体系,探讨SUMO化修饰对TDG活性产生的影响。结果·通过结构解析得到SUMO-1-TDG分子结构。稳定性、活性实验检测发现TDG蛋白熔解温度(Tm)值提高约16℃,催化活性提升约9.70%。结论·SUMO-1分子结合TDG对其进行修饰使得结合位点附近氨基酸形成分子间相互作用,并进一步参与调控TDG蛋白的稳定性及催化活性。
Objective To study the effect of sumoylation on the structure, stability and activity of human thymine DNA glycosylase （TDG）. Methods. Expression and purification systems were established for obtaining SUMO-1-TDG protein with high purity which can be used for crystal screening and activity detection. Structure of SUMO-1-TDG was solved after crystal screening, diffraction data collection and structure analysis. The change of TDG stability led by sumoylation was detected through a protein thermal shift assay. In addition, an activity assay was applied to investigate the effect of sumoylation on the activity of TDG. Results A high-resolution structure of SUMO-1-TDG which could clearly describe the interaction between TDG and SUMO-1 was solved. The melting temperature （Tm） value of SUMO- 1-TDG increased by about 16℃ and the catalytic activity increased by 9.70%, comparing with TDG protein. Conclusion SUMO-1 binds to TDG to modify the intermolecular interaction of amino acids near the binding site, and further participatcs in the regulation of the stability and catalytic activity of TDG protein.
出处
《上海交通大学学报（医学版）》
CAS
CSCD
北大核心
2018年第1期24-29,共6页
Journal of Shanghai Jiao tong University:Medical Science
基金
国家自然科学基金(21572133)~~
